Mussel protein-hyaluronic acid hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a mussel protein-hyaluronic acid hydrogel as well as a preparation method and application thereof, the raw materials of the hydrogel comprise mussel protein and a gelling matrix, the concentration of the mussel protein is 1-100mg / mL, and the concentration of the gelling matrix is 1-100mg / mL; the gel forming matrix is hyaluronic acid modified with a double-bond side chain. The acrylic ester modified hyaluronic acid is used as a gel forming matrix, mussel protein is introduced to form the hydrogel dressing, the mussel protein has good antibacterial performance, oxidation resistance and adhesion performance, and the hyaluronic acid has the effects of promoting cell proliferation and collagen synthesis, moisturizing and resisting bacteria, so that the mussel protein can be used for preparing the hydrogel dressing. And the two functions are combined to serve as a high-quality multifunctional dressing for resisting bacteria, regulating an oxidation environment and promoting repair.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a mussel protein-hyaluronic acid hydrogel and a preparation method and application thereof. Background Art

[0002] Diabetic foot ulcer (DFU) is one of the most common and serious complications of diabetes. DFU occurs in nearly 25% of diabetic patients and usually leads to chronic wounds due to healing disorders. Studies have shown that the risk of death in patients with diabetic foot ulcers is 2.5 times that of diabetic patients without foot ulcers, and more than 50% of diabetic foot ulcers will cause infection. In addition, about 20% of moderate or severe diabetic foot infections will lead to some degree of amputation. DFU is one of the important causes of disability and even death in people with diabetes, which not only reduces the quality of life of patients, but also adds a huge economic burden.

[0003] Normal wound healing is a complex and orderly process, divided into four overlapping stages: hemostasis, inflammation, proliferation and remodeling. However, diabetic wounds have some unique characteristics, and wound repair does not follow the normal process and is unbalanced. In diabetic foot wounds, the hyperglycemic microenvironment is one of the characteristics of diabetic foot ulcers and the main reason for their difficulty in recovery. Clinically, high blood sugar levels at the site of injury not only cause cell damage and vascular lesions, but also provide more nutritional resources for bacterial growth and proliferation, leading to recurrent bacterial infections. In addition, abnormal glucose metabolism inhibits the expression of antioxidants and induces advanced glycation end products, which promote oxidative stress and further accumulate inflammation. At the same time, local hyperglycemia can induce endothelial cell apoptosis, inhibit the migration ability of fibroblasts and keratinocytes, and promote the significant expression of matrix metalloproteinase-1 (MMP-1), thereby limiting proliferation and remodeling processes. This complex microenvironment, such as hyperglycemia, hypoxia, excessive wound exudate, recurrent bacterial infection, accumulation of reactive oxygen species, disordered expression of cytokines and growth factors, increased protease activity, persistent inflammation, and impaired angiogenesis and tissue regeneration, will hinder the healing process and prolong one or more of the four overlapping stages of wound healing, increasing the difficulty of clinically treating diabetic wounds.

[0004] In view of the above characteristics, wound dressings have been widely used in the treatment of diabetic wounds due to their significant advantages, such as protecting wounds, absorbing exudates, regulating moisture, preventing bacterial infection and targeted delivery of drugs to promote healing. However, the diabetic wound environment is complex, and the high-sugar environment not only prolongs the inflammatory period, causes redox imbalance (high reactive oxygen species, Reactive oxygen Species, ROS), reduces the expression of important growth factors, and provides more nutrients for bacterial growth and proliferation. Therefore, there is an urgent need to develop multifunctional wound dressings that can adapt to the complex microenvironment of diabetic wounds. Summary of the invention

[0005] One of the purposes of the present invention is to provide a hydrogel, the raw materials of which include mussel protein and a gelling matrix, the concentration of the mussel protein is 1-100 mg / mL, and the concentration of the gelling matrix is ​​1-100 mg / mL;

[0006] The gel-forming matrix is ​​hyaluronic acid modified with double-bond side chains.

[0007] Furthermore, the hyaluronic acid modified with a double bond side chain is hyaluronic acid modified with acrylate.

[0008] Furthermore, the preparation method of the hyaluronic acid modified with acrylate is: dissolving hyaluronic acid in deionized water, adding methacrylic anhydride after stirring, adjusting the pH of the reaction solution to 8-9, and reacting by stirring in an ice bath to obtain hyaluronic acid modified with acrylate. In a specific embodiment of the present invention, the mass volume ratio of hyaluronic acid and methacrylic anhydride is 0.2g:42.5-255μL.

[0009] The second object of the present invention is to provide a method for preparing the above hydrogel, wherein mussel protein, a gelling matrix and a photoinitiator are added to water, and cross-linked by ultraviolet light to obtain a hydrogel.

[0010] Furthermore, the UV cross-linking time is 1-15 minutes.

[0011] The third object of the present invention is to provide a microneedle patch, wherein the microneedle array of the microneedle patch is made of the above-mentioned mussel protein-hyaluronic acid hydrogel.

[0012] A fourth object of the present invention is to provide the use of the above-mentioned hydrogel or microneedle patch in the preparation of external dressings for diabetic wounds.

[0013] In view of the complex case environment of ulcer wounds in the diabetic group, the present invention designs a mussel protein-hyaluronic acid hydrogel dressing. On the one hand, from the perspective of functional synthesis, mussel protein itself has good antibacterial properties, antioxidant properties, and adhesion properties, and hyaluronic acid has the functions of promoting cell proliferation, promoting collagen synthesis, moisturizing and antibacterial effects. The combination of the two functions can be used as a high-quality antibacterial, oxidative environment regulation, and a multifunctional dressing for repair. On the other hand, from the perspective of the preparation method, acrylate-modified hyaluronic acid is used as a gelling matrix, and mussel protein is introduced to form a hydrogel dressing. The mussel protein-hyaluronic acid hydrogel dressing prepared by the present invention, including the hardness mechanical property changes of the hydrogel formed by hyaluronic acid and mussel protein modified with different grafting rates. In addition, since the microneedle patch can be attached to the skin wound for a long time and stably, and overcomes the traditional mode of administration, such as local application or intradermal injection, the drug cannot be effectively transported to the deep layer, thereby limiting the limitation of its efficacy, therefore, the present invention further makes mussel protein-hyaluronic acid hydrogel into mussel protein drug-loaded microneedles to enhance wound repair performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the hydrogel prepared in Example 1.

[0015] Figure 2 To investigate the hemolytic properties of the hydrogel.

[0016] Figure 3 To investigate the antibacterial properties of the hydrogel against Escherichia coli and Staphylococcus aureus.

[0017] Figure 4 Live-dead staining was used to investigate the effects of hydrogel on L929 cells and HUVEC.

[0018] Figure 5 To investigate the hemostatic ability of hydrogel on mouse tail bleeding.

[0019] Figure 6 It is a mussel protein-hyaluronic acid hydrogel composite microneedle patch.

[0020] Figure 7 This is a photo of the microneedle inserted into the abdominal skin of a mouse. DETAILED DESCRIPTION

[0021] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0024] Example 1

[0025] 1. Directional control of the synthesis of mHA

[0026] In this example, 90 kDa HA is used as an example to synthesize mHA, and the synthesis route is shown in the following formula:

[0027]

[0028] Specifically: 0.2g HA was dissolved in 10mL deionized water and stirred overnight in an ice bath, then 85μL methacrylic anhydride (MA) was added, and the pH was adjusted with 1M NaOH so that the reaction pH was maintained between 8 and 9, and the reaction was stirred in an ice bath for 24h. Thereafter, the reaction solution was added to pre-cooled anhydrous ethanol for precipitation and washed with ethanol 3 times. The obtained product was redissolved in water and placed in a dialysis bag (8-14KDa) for 48h (8 water changes) and then freeze-dried to obtain mHA.

[0029] mHA is dissolved in D 2 O 1 H-NMR (400 MHz, D 2 The grafting rate was calculated by characterizing the mHA with different grafting rates according to the above method, and the addition amount of MA was 255, 170, 85 and 42.5 μL, respectively. The experimental results show that the grafting rates of the products are 65.2%, 30.9%, 16.4% and 10.5%, respectively. Therefore, the grafting rate of mHA can be controlled by controlling the addition ratio of HA and MA, and the synthetic grafting rate of mHA can be controlled directionally through fine regulation.

[0030] 2. Preparation of mussel protein-hyaluronic acid hydrogel

[0031] This system selects mHA with a grafting rate of 16.4% as an example. The mixed system is made by adding 1% mHA, 1% mussel protein and 0.1% Irgacure 2959 into water, and then placing it under a 365nm ultraviolet lamp for 1 minute for crosslinking. The gel prepared by this system is a 1% hydrogel. The obtained hydrogel is as follows Figure 1 shown.

[0032] The performance of the prepared 1% hydrogel was investigated below.

[0033] 1. Investigation of Antioxidant Capacity

[0034] The determination principle is: in an acidic system, Fe 3+-Tripyridine triazine (Fe 3+ -TPTZ) can be reduced by antioxidants to generate blue Fe 2+ -TPTZ, the maximum absorption wavelength is 593nm. Standard curve preparation: 40μmol / mL FeSO 4 The standard solution was diluted with distilled water to 0.1, 0.05, 0.025, 0.0125, 0.00625, and 0.003125 μmol / mL. The standard solution was thoroughly mixed with the detection reagent (total antioxidant capacity (T-AOC) detection kit, Solarbio, BC1315). After reacting for 10 min, the absorbance at 593 nm was measured to calculate the total antioxidant capacity, Fe 2+ The final concentration is the horizontal axis (x), and the antioxidant capacity is the vertical axis (y) to draw a linear equation y = KX + b, and the subsequent measured ΔA is substituted into the equation to obtain x (μmol / mL). 3 +-TPTZ was added with 24 μL of 1% hydrogel solution, and the total volume of the reaction system was 204 μL. The distilled water group was set as A. 空白 , sample group A 测定 After 10 minutes of reaction, take 200 μL and measure the absorbance at 593 nm. ΔA=A 测定 -A 空白 , the total antioxidant capacity was calculated as follows: total antioxidant capacity (μmol / mL) = 34 × x. The results showed that the hydrogel had significant antioxidant properties.

[0035] 2. Hemolytic performance test

[0036] Whole blood was collected from the retro-orbital venous plexus of rats, collected in a centrifuge tube containing sodium heparin (10 mg / mL), and centrifuged at 2000 rpm for 10 min at 4°C to collect red blood cells, and then the red blood cells were repeatedly washed with 0.9% NaCl solution by centrifugation (2000 rpm, 5 min) until there was no red in the supernatant. The purified red blood cells were diluted with 0.9% NaCl to obtain a red blood cell suspension (2%, v / v), and then 100 μL of the red blood cell suspension was incubated with 100 μL of 1% hydrogel, with Triton X100 and PBS used as positive and negative controls, respectively. After incubation at 37°C for 3 hours, the supernatant was centrifuged and added to a 96-well plate, and the absorbance at a wavelength of 570 nm was detected. The hemolysis rate was calculated as: HR (%) = [(Agel-As) / (At-As)] × 100%. Agel, At and As represent the absorbance of each sample, positive control and negative control at 570 nm, respectively. Figure 2 As shown, the experimental results show that the hemolysis rate is less than 5%, proving that the material does not cause hemolysis.

[0037] 3. Antibacterial performance test

[0038] Weigh 1g of peptone, 0.5g of yeast powder, 1g of NaCl, and 2g of agar powder, dissolve them in 100mL of deionized water, mix well, and sterilize at 121℃ for 20min. Then pour it into a 100mm cell culture dish while it is hot in the clean bench, and let the excess water on the surface solidify and air-dry for use. First, take out the Escherichia coli (E.Coli) and methicillin-resistant Staphylococcus aureus (MRSA) frozen in a -80℃ refrigerator and add them to LB medium. Then place the culture bottle in a constant temperature shaker at 37℃ and 180rpm for overnight culture. The next day, collect the bacterial solution during the exponential growth phase. The bacterial concentration was determined by measuring the OD of the bacterial solution at a wavelength of 600nm. Before using the hydrogel for antibacterial experiments, the OD600 value of the bacterial stock solution was readjusted to 0.1, which is consistent with the concentrations of Staphylococcus aureus and Escherichia coli obtained based on the colony counting method, which are 2×10 8 and 1×10 8 CFU / mL. 50 μL of bacterial stock solution dilution (1.0×10 6 CFU / mL) and 50μL 1% hydrogel were added to a 96-well plate, and a well plate without hydrogel but with bacterial solution was used as a control, and incubated at 37°C for 2h. The solution in each well was diluted 1000 times, 100ml of bacterial solution was evenly spread on the LB agar plate, inverted and cultured at 37°C, and the colonies on the LB agar plate were counted after 16-18h. The results are shown in Figure 3 As shown, the results show that the hydrogel has good antibacterial activity against both Escherichia coli and methicillin-resistant Staphylococcus aureus.

[0039] 4. In vitro cell safety test

[0040] Human umbilical vein endothelial cells HUVEC and mouse fibroblasts L929 were used to investigate the safety of the hydrogel using MTT technique and live-dead staining technique. HUVEC and L929 were taken out of the liquid nitrogen tank, and the cells were immediately placed in a 37°C water bath, and slowly shaken until the cell fluid was completely dissolved. The thawed cells were placed in an ultra-clean bench, blown evenly with a pipette, transferred to a centrifuge tube, complete culture medium was added, centrifuged and the supernatant was discarded, and then the culture medium was added to blow evenly, and finally all of them were transferred to a culture bottle and placed in a 37°C, 5% CO 2 When the cell viability reaches more than 95%, subsequent experiments can be carried out.

[0041] First, the effect of hydrogel on the viability of HUVEC and L929 cells was studied using the thiazolyl blue colorimetric method (Methyl thiazolyl tetrazolium, MTT). The two logarithmically growing cell suspensions were diluted and 1 mL was added to each well of a 24-well plate. After the cells were cultured in the incubator for 24 hours, the complete medium was replaced with an incomplete medium (DMEM high-glucose medium with 1% penicillin and 1% streptomycin and RPMI-1640 medium with 1% penicillin and 1% streptomycin). In the hydrogel group, 50 μL of 1% hydrogel was added to the Transwell chamber for a continuous co-culture of 24 hours, and then 100 μL of MTT solution (5 mg / mL) was added to each well, and then the well plate was returned to the incubator for another 4 hours. After the incubation, the culture medium was removed, 750 mL of DMSO was added to each well, and the absorbance at a wavelength of 570 nm was immediately detected using an ELISA reader. The cell survival rate was calculated as: (ODgel-OD0) / (ODcon-OD0)×100%, where ODgel represents the hydrogel group, ODcon represents the control group, and OD0 represents the blank group. The results showed that the survival rate of the hydrogel after co-incubation with HUVEC cells and L929 cells was as high as over 90%, and the hydrogel matrix had almost no toxic effect on HUVEC cells and L929, and had good biocompatibility.

[0042] In order to further simulate the effect of gel on cells in vivo, the Transwell method was used to investigate. The cell suspensions of logarithmically growing HUVEC and L929 cells were inoculated in 24-well plates. After the cells were cultured in the incubator for 24 hours, the gel was placed on the upper layer of the Tranwell chamber (same as above) and co-cultured with the cells as the gel group, and PBS was added in the same way as the control group. After 24 hours of culture, the cells were stained with the Calcein-Am / PI double staining kit, and finally observed and photographed under an inverted fluorescence microscope. The results are shown in the figure. Figure 4 As shown, the results showed that the hydrogel group had basically no red fluorescence representing dead cells, similar to the control group, and the cell morphology did not change, further indicating that the hydrogel was basically non-toxic.

[0043] 5. Investigation of ROS scavenging ability

[0044] L929 cells growing in logarithmic growth were taken at 3×10 5The cells were inoculated into six-well plates and cultured for 24 hours. After co-culture with incomplete culture medium, incomplete culture medium containing 0.3mM hydrogen peroxide, and hydrogel group in Transwell chambers, 100μL of 1% hydrogel was added and co-cultured for 24 hours. Then, the cells were gently rinsed with PBS, and 0.5mL of DCFH-DA probe (PBS diluted 1000 times to 10μM) was added. The cells were incubated at 37℃ in the dark for 30 minutes. The cells were gently rinsed with PBS three times, and finally 300μL of PBS was added and placed under a fluorescence microscope for observation and photography. The results showed that the green fluorescence of intracellular ROS was significantly enhanced after the addition of hydrogen peroxide, and the enhancement of intracellular ROS caused by hydrogen peroxide was significantly eliminated after the addition of hydrogel.

[0045] 6. Hemostatic performance test

[0046] Female ICR mice were fed for 1 week before surgery to adapt to the environment. During surgery, the mice under general anesthesia were fixed on the operating board, and a pre-weighed filter paper was placed under the tail. The scalpel quickly cut off 1 / 3 of the mouse's tail, and the timing was started. At the same time, 100 μL of hydrogel (1%) was applied to the bleeding site. The wounds in the control group did not receive any treatment. An electronic device was used to record the bleeding time of the wound. The blood-absorbing filter paper was weighed and compared with the bleeding amount in the control group. The experimental results are shown in the figure below. Figure 5 As shown, the hydrogel dressing can significantly shorten the hemostasis time, reduce the amount of bleeding, and has good hemostatic properties.

[0047] Example 2

[0048] Preparation of microneedle dressings with mussel protein-hyaluronic acid hydrogel

[0049] A mixed solution of 1% mussel protein, 3% mHA, 3% N,N-methylenebisacrylamide (MBA) and 0.1% Irgacure 2959 was added to the PDMS mold reservoir to prepare the needle tip, and the base was prepared with a mixed solution of 4% mHA, 4% MBA and 0.1% Irgacure 2959. The dried microneedle patch was separated from the PDMS mold, and the needle body was cross-linked under UV for 1 min.

[0050] like Figure 6 As shown, the patch consists of 100 (10×10) microneedles arranged in a 9×9 mm 2 Fluorescence images taken under a microscope show that the protein is evenly distributed in the patch.

[0051] In order to investigate the skin insertion performance of the prepared microneedle patch, the microneedle patch was pressed on the peeled mouse abdominal skin with the thumb for 5 minutes, then the microneedle base was removed, the residual sample on the mouse skin was wiped clean, and it was stained with 1% trypan blue for 5 minutes, the residual trypan blue on the skin was washed off, and finally photos were taken. The experimental results are shown in Figure 2. Figure 7 As shown, the prepared microneedle patch was able to successfully pierce the skin and form a channel with relatively uniform pore size, proving that the prepared microneedle patch has sufficient mechanical strength for transdermal treatment.

Claims

1. A hydrogel, characterized in that: The raw materials include mussel protein and a gelling matrix, the concentration of the mussel protein is 1-100 mg / mL, and the concentration of the gelling matrix is ​​1-100 mg / mL; The gel-forming matrix is ​​hyaluronic acid modified with double-bond side chains.

2. The hydrogel according to claim 1, characterized in that The hyaluronic acid modified with a double bond side chain is hyaluronic acid modified with acrylate.

3. The hydrogel according to claim 2, characterized in that The preparation method of the hyaluronic acid modified with acrylate is as follows: dissolving hyaluronic acid in deionized water, adding methacrylic anhydride after stirring, adjusting the pH of the reaction solution to 8-9, and reacting by stirring in an ice bath to obtain the hyaluronic acid modified with acrylate.

4. The hydrogel according to claim 3, characterized in that The mass volume ratio of the hyaluronic acid and methacrylic anhydride is 0.2 g:42.5-255 μL.

5. The method for preparing the hydrogel according to any one of claims 1 to 4, characterized in that: Mussel protein, gelling matrix and photoinitiator are added into water, and cross-linked by ultraviolet light to obtain hydrogel dressing.

6. The method for preparing the hydrogel according to claim 5, characterized in that: The time of the ultraviolet light cross-linking is 1-15 min.

7. A microneedle patch, characterized in that: The microneedle array of the microneedle patch is made of the mussel protein-hyaluronic acid hydrogel according to any one of claims 1 to 4.

8. Use of the hydrogel according to any one of claims 1 to 4 or the microneedle patch according to claim 7 in the preparation of a diabetic wound dressing.

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